PCR-based screening for insertional mutants.

In Arabidopsis, where homologous recombination is highly inefficient, insertional mutagenesis has provided a great alternative means for identifying loss-of-function mutants in genes of interest. Many collections of T-DNA and transposon-tagged plants have been generated and several have been made available to the research community. With recent sequencing of the insertion sites and the creation of public databases, identification of knockouts in most of the genes of interest has become a matter of a few minutes. However, because of the random selection of the insertion lines to be sequenced, knockouts of the remaining several thousand genes may not be easily found by the mere continuation of these types of approaches. Therefore, alternative methods such as polymerase chain reaction (PCR)-based screening of pooled genomic DNA from mutant lines using a combination of tag- and gene-specific primers, once again, are becoming valuable for obtaining loss-of-function alleles in the genes "missed" by the sequencing projects. In this chapter, we provide a detailed description of the design and implementation of a PCR-based screen for insertional knockouts using T-DNA-mutagenized lines as an example.